地黄寡糖对3T3-L1脂肪细胞增殖及胰岛素抵抗的作用

目的：探讨地黄寡糖对脂肪细胞增殖和胰岛素抵抗的影响。方法：培养3T3-L1前脂肪细胞,用四甲基偶氮唑盐（MTT）方法检测3T3-L1前脂肪细胞及脂肪细胞的增殖情况,同时采用地塞米松诱导3T3-L1脂肪细胞建立胰岛素抵抗模型,检测地黄寡糖对细胞培养基中葡萄糖浓度的影响。结果：在DMEM高糖培养基中,地黄寡糖可促进3T3-L1前脂肪细胞增殖,抑制3T3-L1脂肪细胞增殖,作用呈明显量效关系；使3T3-L1前脂肪细胞及3T3-L1脂肪细胞葡萄糖消耗量增加,呈明显量效关系；地黄寡糖能明显增加胰岛素抵抗3T3-L1脂肪细胞培养基中的葡萄糖消耗量,增强对胰岛素的敏感性。结论：地黄寡糖可以促进前脂肪细胞的增殖,抑制脂肪细胞的增殖,地黄寡糖对地塞米松诱导的3T3-L1脂肪细胞胰岛素抵抗具有明显的改善作用。     

小檗碱对3T3-L1脂肪细胞增殖及糖代谢的影响

目的:研究小檗碱对脂肪细胞增殖、糖代谢及地塞米松诱导3T3-L1脂肪细胞中葡萄糖转运蛋白4(GLUT4)mRNA表达的影响。方法:用四甲基偶氮唑盐(MTT)方法检测3T3-L1前脂肪细胞及脂肪细胞的增殖情况,检测小檗碱对3T3-L1前脂肪细胞及脂肪细胞培养基中葡萄糖浓度的影响;采用地塞米松诱导胰岛素抵抗细胞模型,分别给予罗格列酮、小檗碱进行干预,用RT-PCR检测GLUT4 mRNA的表达。结果:在DMEM高糖培养基中,小檗碱可促进3T3-L1前脂肪细胞增殖,抑制3T3-L1脂肪细胞增殖,作用呈明显量效关系;使3T3-L1前脂肪细胞及3T3-L1脂肪细胞葡萄糖消耗量增加,呈明显量效关系,同时小檗碱组GLUT4 mRNA的表达升高。结论:小檗碱可以显著促进前脂肪细胞的增殖,抑制脂肪细胞的增殖;同时具有显著的降糖作用。且小檗碱能上调脂肪细胞GLUT4 mRNA的表达,这提示小檗碱改善胰岛素抵抗的作用机制可能与罗格列酮不同。     

小檗碱对白介素-6诱导胰岛素抵抗 3T3-L1脂肪细胞脂联素表达的影响

目的:观察小檗碱对白介素-6(IL-6)诱导胰岛素抵抗的3T3-L1脂肪细胞脂联素表达的影响。方法:选用IL-6诱导3T3-L1脂肪细胞胰岛素抵抗(IR)模型。以20μg·L-1IL-6培养48 h,将3T3-L1脂肪细胞随机分为正常对照组、模型组、吡格列酮组(50μmol.L-1)和小檗碱高、中、低剂量组(10,20,50μmol.L-1),以葡萄糖氧化酶法测定葡萄糖消耗量,观察小檗碱对脂肪细胞葡萄糖摄取的影响,鉴定IR模型;采用实时荧光定量PCR技术测定脂肪细胞脂联素基因mRNA水平。结果:模型组葡萄糖消耗量及脂联素基因表达水平与正常对照组比较,均显著降低(P<0.05);小檗碱高、中、低剂量组及吡格列酮组均能显著增加葡萄糖消耗量及脂联素基因表达水平(P<0.05)。结论:小檗碱可增加白介素-6诱导胰岛素抵抗的3T3-L1脂肪细胞脂联素基因mRNA的表达,改善胰岛素抵抗状态。     

毛蕊异黄酮葡萄糖苷与葛根素及其配伍对3T3-L1前脂肪细胞胰岛素抵抗细胞模型的影响

目的:研究毛蕊异黄酮葡萄糖苷与葛根素及其配伍对3T3-L1前脂肪细胞增殖分化和胰岛素抵抗(IR)脂肪细胞糖脂代谢的影响。方法:采用MTT法检测毛蕊异黄酮葡萄糖苷与葛根素及其配伍对3T3-L1前脂肪细胞增殖的影响。采用鸡尾酒式诱导脂肪细胞分化,油红O染色法鉴定脂肪细胞分化情况,地塞米松诱导脂肪细胞建立IR模型。分别给予毛蕊异黄酮葡萄糖苷、葛根素、毛蕊异黄酮葡萄糖苷+葛根素、吡咯列酮进行干预,药物作用48h后以葡萄糖氧化酶-过氧化物酶法(GOD-POD法)检测培养液中葡萄糖消耗量,甘油磷酸氧化酶-过氧化物酶法(GPO-PAP法)检测细胞内TG的含量。结果:成功建立3T3-L1脂肪细胞IR模型。与正常对照组比较,毛蕊异黄酮葡萄糖苷(20、200、2 000μmol/L)与葛根素(0.1、1、10μmol/L)及毛蕊异黄酮葡萄糖苷+葛根素配伍(20μmol/L+0.1μmol/L、200μmol/L+1μmol/L、2 000μmol/L+10μmol/L)组均能明显促进3T3-L1细胞的增殖。毛蕊异黄酮葡萄糖苷(200μmol/L)、葛根素(1μmol/L)、毛蕊异黄酮葡萄糖苷+葛根素(200μmol/L+1μmol/L)组作用48h后对细胞的增殖作用与吡咯列酮组最接近,因此选择该药物浓度与作用时间进行后续实验。与模型组相比,毛蕊异黄酮葡萄糖苷(200μmol/L)与葛根素(1μmol/L)及毛蕊异黄酮葡萄糖苷+葛根素(200μmol/L+1μmol/L)配伍组均可显著增加IR脂肪细胞培养液的葡萄糖消耗量,明显降低细胞中TG的含量,毛蕊异黄酮葡萄糖苷与葛根素配伍组效果显著优于单药组。结论:毛蕊异黄酮葡萄糖苷、葛根素及其配伍能明显促进3T3-L1前脂肪细胞的增殖,且可明显提高IR脂肪细胞的葡萄糖消耗量,降低TG含量,可通过调节糖代谢和脂代谢改善IR。     

芦丁对3T3-L1前脂肪细胞分化的作用

目的探索芦丁对3T3-L1前脂肪细胞分化的影响。方法采用3T3-L1前脂肪细胞分化模型和油红O染色法,选用罗格列酮作为阳性对照药,观察芦丁对3T3-L1前脂肪细胞分化的作用,于510 nm波长下测定芦丁对脂含量的影响。利用流式细胞仪测定芦丁对分化的脂肪细胞吸收葡萄糖的影响。结果芦丁在0.5μmol/L和1μmol/L两个浓度下均能促进3T3-L1前脂肪细胞分化过程,提高脂含量,并促进了葡萄糖吸收。结论芦丁能够促进3T3-L1前脂肪细胞分化,并促进了分化的脂肪细胞吸收葡萄糖。     

黄连改善胰岛素抵抗药效物质基础研究

目的:从细胞途径多方面观察黄连药材、部位及其成分等对3T3-L1前脂肪细胞诱导分化及脂肪细胞胰岛素抵抗的影响,探索黄连改善胰岛素抵抗的药效物质基础。方法:培养3T3-L1前脂肪细胞,采用地塞米松、3-异丁基-1-甲基黄嘌呤和胰岛素共同诱导3T3-L1前脂肪细胞分化为脂肪细胞,并建立胰岛素抵抗模型。依据黄连传统用药经验,观察黄连"药材-部位-生物碱成分"对脂肪细胞分化的影响,对胰岛素抵抗模型细胞培养液中葡萄糖消耗的影响。结果:除非生物碱部位在6.0μg.L-1具有明显促进分化作用外,其余各组无论黄连水提物、不同提取部位还是不同生物碱成分均能明显抑制3T3-L1前脂肪细胞的分化,其中单体成分盐酸黄连碱在浓度16.5μmol.L-1时抑制作用最为明显;同时,各组也均能降低培养液中葡萄糖的含量,提高葡萄糖的利用率,改善胰岛素抵抗,作用与马来酸罗格列酮相似,其中单体成分盐酸药根碱在浓度10.5μmol.L-1时改善作用明显优于其余各成分。结论:黄连具有明显改善胰岛素抵抗,抑制前脂肪细胞分化的作用,其疗效可能是各成分协同作用的结果;同时其抑制分化作用也提示黄连在增加细胞对葡萄糖摄取的同时不会引起脂肪的聚集而造成体重增加,这对防治与胰岛素抵抗相关的代谢综合征或并发症具有一定临床意义。     

胰岛素抵抗3T3-L1脂肪细胞模型的建立及鉴定

目的建立胰岛素抵抗3T3-L1脂肪细胞模型.方法培养3T3-L1前脂肪细胞,诱导分化为脂肪细胞,采用地塞米松诱导建立3T3-L1脂肪细胞胰岛素抵抗模型,检测不同时间段细胞培养基中葡萄糖浓度的变化,同时运用RT-PCR技术检测抵抗素(Resistin)基因的表达.结果随着时间的变化培养基中的葡萄糖浓度逐渐降低,在96 h达到胰岛素抵抗的最高峰,此时模型组的Resistin基因表达较空白组升高了约3倍.结论将3T3-L1脂肪细胞置于1μmol/L地塞米松环境中24 h,该细胞对胰岛素的作用产生抵抗,此胰岛素抵抗状态可以维持216 h.     

肿瘤坏死因子-α诱导3T3-L1脂肪细胞胰岛素抵抗模型的建立

目的:应用肿瘤坏死因子d(TNF-α)诱导3T3-L1脂肪细胞,探讨建立可靠胰岛素抵抗(IR)细胞模型的方法.方法:3T3-L1前脂肪细胞经3-异丁基-1-甲基黄嘌呤、地塞米松、胰岛素诱导分化成3T3-L1脂肪细胞,将其与20,10,5μg·L-1TNF-α共孵育,100 nmol·L-1胰岛素作用30 min刺激脂肪细胞糖转运.以葡萄糖氧化酶法测定培养基上清液葡萄糖含量,观察TNF-α对脂肪细胞糖摄取的影响,鉴定IR模型.结果:TNF-α抑制胰岛素诱导前、后的脂肪细胞糖转运,抑制作用呈剂量依赖性,其中20 μg·L-1TNF-α的抑制率分别为79.2％和81.4％(P＜0.05).结论:肿瘤坏死因子α可诱导3T3-L1脂肪细胞产生IR,这种细胞模型简便、可靠.     

3’-羟基葛根素对脂肪细胞3T3-L1胰岛素抵抗的影响及其机制研究

目的探讨3′-羟基葛根素改善胰岛素抵抗的作用及其机制。方法采用MTT法检测3′-羟基葛根素对3T3-L1前脂肪细胞增殖的影响;油红O染色法检测其对3T3-L1前脂肪细胞分化的影响。以地塞米松诱导分化成熟的脂肪细胞,建立胰岛素抵抗模型,分别采用葡萄糖氧化酶法和比色法检测细胞培养上清液中葡萄糖消耗量和游离脂肪酸(FFA)生成量;实时荧光定量PCR法分析脂肪细胞中过氧化物酶体增殖物激活受体γ(peroxysome proliferator-activated receptor gamma,PPARγ)和蛋白酪氨酸磷酸酶1B(protein tyrosine phosphatase 1B,PTP1B)的基因表达。采用嵌合蛋白基因试验检测3′-羟基葛根素的PPARγ配体结合活性和比色法检测其对PTP1B酶活性的影响。结果与溶媒对照组相比,1~10μmol/L 3′-羟基葛根素显著促进3T3-L1前脂肪细胞增殖及细胞分化(P<0.05、0.01)。与模型组相比,无论在基础状态还是胰岛素刺激状态,3′-羟基葛根素均能显著增加胰岛素抵抗脂肪细胞葡萄糖利用率、减少FFA的产生(P<0.05、0.01);同时显著上调胰岛素抵抗脂肪细胞PPARγ基因的表达,但对PTP1B基因表达无明显影响(P>0.05)。与溶媒对照组相比,3′-羟基葛根素在0.1、10.0μmol/L时能对PPARγ产生激活作用(P<0.05、0.01),但对PTP1B酶活性没有明显抑制作用(P>0.05)。结论 3′-羟基葛根素能促进胰岛素抵抗脂肪细胞葡萄糖利用、抑制FFA产生,从而改善胰岛素抵抗,其机制可能与上调PPARγ基因表达有关。     

葛根素对前脂细胞分化及胰岛素抵抗模型糖摄取的影响

目的探讨葛根素对3T3-L1前脂细胞分化及胰岛素抵抗模型糖摄取的影响。方法 (1)实验分为葛根素3,10,30,100,300μmol/L组、空白组、罗格列酮20μmol/L组,各加药组分别给予相应浓度药物对3T3-L1前脂细胞进行干预,空白组常规培养,采用油红O染色法检测各组3T3-L1前脂细胞分化情况。(2)实验分为葛根素3,10,30,100,300μmol/L组、空白组、罗格列酮20μmol/L组,除空白组外,其余各组应用地塞米松对3T3-L1前脂细胞诱导进行胰岛素抵抗模型的建立,然后分别给予相应浓度药物对3T3-L1前脂细胞进行干预,空白组常规培养,检测各组细胞葡萄糖摄取量。结果葛根素3,10μmol/L组细胞分化成脂的相对变化率与空白组比较差异均无统计学意义(P均>0.05),葛根素30,100,300μmol/L组和罗格列酮20μmol/L组细胞分化成脂的相对变化率均明显高于空白组(P均<0.05);葛根素3,10μmol/L组胰岛素抵抗模型3T3-L1前脂细胞葡萄糖摄取相对变化率与空白组比较差异均无统计学意义(P均>0.05),葛根素30,100,300μmol/L组和罗格列酮20μmol/L组胰岛素抵抗模型3T3-L1前脂细胞葡萄糖摄取相对变化率均明显高于空白组(P均<0.05)。结论低浓度葛根素对3T3-L1前脂细胞影响较小,高浓度的葛根素能够促进3T3-L1前脂细胞分化及提高胰岛素抵抗模型3T3-L1前脂细胞葡萄糖摄取率。     

催产素对3T3-L1脂肪细胞糖脂代谢的影响

目的观察催产素对3T3-L1脂肪细胞糖脂代谢的影响。方法 3T3-L1前脂肪细胞体外培养,并诱导其分化成熟为脂肪细胞。研究催产素对脂肪细胞葡萄糖消耗量以及三酰甘油、游离脂肪酸和甘油的影响。采用实时荧光定量PCR法检测糖脂代谢相关基因GLUT-1、GLUT-4、ATGT、HSL的mRNA表达。结果与对照组比较,催产素20、50、100μg/m L组葡萄糖消耗量有所增加,且表现出剂量相关。催产素组较对照组的三酰甘油降低,而甘油和游离脂肪酸增高。催产素50μg/m L组中脂代谢相关基因HSL表达明显高于对照组,糖代谢相关基因GLUT-4 mRNA表达水平增加。结论催产素处理可减少3T3-L1细胞脂质合成、增加脂质分解作用,并可明显改善脂质积聚。     

Interaction of baicalin with berberine for glucose uptake in 3T3-L1 adipocytes and HepG2 hepatocytes

Ethnopharmacological relevance

Baicalin and berberine are important coexisting constituents of the combination of Radix Scutellariae and Rhizoma Coptidis, known as scutellaria–coptis herb couple (SC), which has heat clearing and detoxifying effects. The aims of the present study were to investigate the effects of the combination of baicalin+berberine on glucose uptake in 3T3-L1 adipocytes or HepG2 cells.

Materials and methods

Insulin-resistant adipocytes and hepatocytes models were established. Glucose consumption was assayed to evaluate the effects of berberine, baicalin, and berberine+baicalin on glucose uptake, and the interaction of baicalin with berberine for glucose uptake was evaluated in 3T3-L1 adipocytes or HepG2 cells. Moreover, the effects of baicalin on the dose–effect relationship of berberine for glucose uptake was also evaluated in 3T3-L1 adipocytes.

Results

The results of the present study demonstrated that berberine increased glucose consumption in 3T3-L1 adipocytes and HepG2 hepatocytes in a dose-dependent manner. In contrast, statistical analyses indicated that baicalin (in doses up to 100 μmol/L) produced no obvious effect. The effect of berberine+baicalin on glucose uptake was better than that of berberine or baicalin alone, which indicated that berberine and baicalin had the trend of synergetic effect on glucose uptake. Furthermore, these results showed that the synergistic effect occurred in a specific dose range, while the antagonistic effect was present in another dose range in the presence of 10 μmol/L baicalin. Interestingly, the entire dose–response curves of berberine shifted down in the presence of 100 μmol/L baicalin, and baicalin antagonised the effect of berberine on glucose uptake in 3T3-L1 adipocytes.

Conclusions

The results of the present study showed that berberine dose-dependently increased glucose consumption in 3T3-L1 adipocytes and HepG2 hepatocytes. Furthermore, interaction of baicalin with berberine was additive at low doses of baicalin and antagonistic at higher baicalin doses. Thus, it is possible that baicalin is a partial agonist. These results provided a basis for the study of the TCM compatibility mechanism and a new insight into the application for Gegen Qinlian Decoction (GGQLD) or SC in the clinic.     

3,4-Oxo-isopropylidene-shikimic acid promotes adiopkine expression during murine 3T3-L1 fibroblast differentiation into adipocytes

Objective3,4-Oxo-isopropylidene-shikimic acid (ISA), a derivative of shikimic acid, has exhibited ameliorative effect on cognitive impairment in experimental animal models of dementia. This study investigated the effect of ISA on lipid accumulation and adipokine secretion during differentiation of 3T3-L1 fibroblasts to adipocytes.Methods3T3-L1 cells were cultured and treated with ISA (50–800 μM) from days 3–8. Lipid accumulation and triglyceride content were measured. Gene expression of adipokines (adiponectin, leptin, and resistin), CCAAT/enhancer binding protein (C/EBP) β, C/EBP α and peroxisome proliferator-activated receptor γ (PPAR γ) and PPAR target genes, including adipocyte fatty acid binding protein (aP2) and fatty acid synthase (FAS) were investigated.ResultsISA promoted 3T3-L1 fibroblast differentiation to adipocytes and increased triglyceride content by 26%. On mechanistic levels, ISA increased expressions of C/EBP β, PPAR γ, C/EBP α, aP2 and FAS. Moreover, ISA stimulated expressions of adipokines secreted by adipocytes, including adiponectin, leptin, and resistin.ConclusionsThese findings demonstrated that ISA promoted adipogenesis by up-regulating expressions of C/EBP β, PPAR γ, C/EBP α, aP2 and FAS, and also stimulated adipokines during adipocyte differentiation. Further study should clarify the relationship between stimulation of adipokines and cognitive enhancing effect of ISA.     

小檗碱对3T3-L1前脂肪细胞增殖及分化相关基因PPARγ、C/EBPα mRNA和蛋白表达的影响

目的 探讨小檗碱对脂肪细胞增殖、分化的影响及其机制。     

胡柚果肉不同极性提取物对3 T3-L1脂肪细胞葡萄糖摄取率、白细胞介素-6及游离脂肪酸的影响

目的：用不同极性的溶剂对胡柚果肉进行依次分离,得到5个不同极性组分,将不同极性组分进行促进3 T3-L1脂肪细胞葡萄糖消耗、降低白细胞介素-6和游离脂肪酸分泌的活性组分的筛选及其机制的研究。方法：采用溶剂法将胡柚果肉分离成5个不同极性的组分;诱导分化3 T3-L1脂肪细胞,成熟后添加不同浓度的提取组分作用48 h,检测细胞培养液中葡萄糖、细胞白介素-6和游离脂肪酸含量,比较加药培养前后的葡萄糖、细胞白介素-6和游离脂肪酸含量,筛选出具有促进3 T3-L1脂肪细胞葡萄糖消耗、降低白细胞介素-6和游离脂肪酸分泌的活性组分。结果：胡柚果肉中极性组分占（88.96±2．34）％,而非极性组分的含量较少,胡柚果肉石油醚组分、二氯甲烷组分、乙酸乙酯组分和水组分均能显著促进脂肪细胞对葡萄糖的摄取,二氯甲烷组分、乙酸乙酯组分、正丁醇组分和水组分能够显著降低脂肪细胞白介素-6和游离脂肪酸的水平。结论：胡柚果肉可通过促进3 T3-L1脂肪细胞对葡萄糖的消耗,降低细胞因子白细胞介素-6和游离脂肪酸水平调节血糖。     

胰岛素抵抗3T3-L1脂肪细胞模型及中药有效成分的干预作用

高糖高胰岛素、游离脂肪酸、炎症因子、地塞米松、金属铬、雌激素、葡糖胺等均可诱导3T3-L1脂肪细胞建立胰岛素抵抗模型,且具有稳定、可靠、易于重复等优点。目前已证实小檗碱+梓醇、小檗碱、熊果酸、西洋参茎叶总皂苷及其他活性成分、蒲黄总黄酮、灵芝多糖、附子多糖、地黄寡糖、积雪草酸、白藜芦醇、葛根素等中药有效成分具有改善胰岛素抵抗及调节糖代谢作用。     

Water extracts from Momordica charantia increase glucose uptake and adiponectin secretion in 3T3-L1 adipose cells

To examine the effects of Momordica charantia on glucose uptake and adiponectin secretion in adipose cells, 3T3-L1 adipocytes were treated with three concentrations (0.2, 0.3 and 0.4mg/ml) of water and ethanol extracts of Momordica charantia fruit and seeds alone and in combination with either 0.5nM or 50nM insulin. The treatment combination of 0.2mg/ml water extract and 0.5nM insulin was associated with significant (p<0.05) increases in glucose uptake (61%) and adiponectin secretion (75%) over control levels. The ethanol extract was not associated with an increase in glucose uptake; however, a dose-dependent decrease in basal glucose uptake and insulin-mediated glucose uptake was observed with the ethanol extract in combination with 50nM insulin. In the absence of insulin, no effects on glucose uptake were observed in adipocytes exposed to the water extracts whereas the highest concentration (0.4mg/ml) of the ethanol extract was associated with a significant (p<0.05) decrease in glucose uptake relative to controls. The present results indicate that water-soluble component(s) in Momordica charantia enhance the glucose uptake at sub-optimal concentrations of insulin in 3T3-L1 adipocytes, which is accompanied by and may be a result of increased adiponectin secretion from the 3T3-L1 adipocytes.